Ovarian follicular growth in the rat is dependent on hormone-induced changes in granulosa cell function. To study the changes in, and factors regulating, expression of mRNAs encoding various granulosa cell proteins cDNA and genomic clones for aromatase cytochrome P450 (CYP19) and RIIBeta, the regulatory subunit of type IIBeta cAMP-dependent protein kinase have been isolated. The marked increase in expression of aromatase and RIIBeta mRNAs associated with the development of preovulatory follicles in vivo and in granulosa cells cultured in the presence of FSH and steroids led us to propose five years ago that these genes might share common cis-acting DNA elements and trans-acting factors. However, sequence analysis and functional characterization of isolated promoter and 5' flanking DNA have revealed major differences in the structure of these two genes. Using transient transfection assays to map functional domains of the promoters and electrophoretic mobility shift assays to identify nuclear factor binding sites within the cis- acting DNA, two regions in the aromatase promoter have been identified. One of these binds a recently isolated orphan member of the steroid/thyroid hormone superfamily. Within the RIIBeta promoter, a complex binding region has been identified but the proteins binding to these sequences remain to be determined. Furthermore, recent evidence suggests that tyrosine kinases, C-kinases and a novel set of hormone- inducible kinases (which are highly expressed in the ovary) may also regulate aromatase and RIIBeta expression at specific stages of development. Specifically, serum/glucocorticoid-regulated kinase (sgk) is rapidly induced by FSH in cultured granulosa cells and the appearance of sgk transcripts precedes the induction of aromatase mRNA. Lastly, although estradiol has long been known to enhance the induction by FSH of genes in granulosa cells, the molecular basis for the effects of estradiol in these cells remains undefined. The development of new technologies, in particular the application of differential display PCR, (DD-PCR) to identify unique genes provides the opportunity to identify and characterize genes regulated in granulosa cells by estradiol. Based on these considerations, the Specific Aims of this proposal are to: 1. Determine the functional regions/factors regulating expression of the aromatase and RIIBeta genes by using in vitro and in vivo approaches. 2. Analyze kinase signaling pathways involved in granulosa cell differentiation; including a set of novel inducible kinases, one of which (sgk) is regulated by FSH as an immediate-early gene. 3. Determine estradiol-regulated function(s) in differentiating granulosa cells by characterizing changes in ER during development and by identifying genes induced by estradiol in granulosa cells using DP-PCR.